Elizabeth M. Kenny

Therapies targeting lipid peroxidation in traumatic brain injury

Lipid peroxidation can be broadly defined as the process of inserting a hydroperoxy group into a lipid. Polyunsaturated fatty acids present in the phospholipids are often the targets for peroxidation. Phospholipids are indispensable for normal structure of membranes. The other important function of phospholipids stems from their role as a source of lipid mediators - oxygenated free fatty acids that are derived from lipid peroxidation. In the CNS, excessive accumulation of either oxidized phospholipids or oxygenated free fatty acids may be associated with damage occurring during acute brain injury and subsequent inflammatory responses. There is a growing body of evidence that lipid peroxidation occurs after severe traumatic brain injury in humans and correlates with the injury severity and mortality. Identification of the products and sources of lipid peroxidation and its enzymatic or non-enzymatic nature is essential for the design of mechanism-based therapies. Recent progress in mass spectrometry-based lipidomics/oxidative lipidomics offers remarkable opportunities for quantitative characterization of lipid peroxidation products, providing guidance for targeted development of specific therapeutic modalities. In this review, we critically evaluate previous attempts to use non-specific antioxidants as neuroprotectors and emphasize new approaches based on recent breakthroughs in understanding of enzymatic mechanisms of lipid peroxidation associated with specific death pathways, particularly apoptosis. We also emphasize the role of different phospholipases (calcium-dependent and -independent) in hydrolysis of peroxidized phospholipids and generation of pro- and anti-inflammatory lipid mediators. This article is part of a Special Issue entitled SI:Brain injury and recovery.

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

Mechanistic characterization of nitrite‐mediated neuroprotection after experimental cardiac arrest

Nitrite acts as an ischemic reservoir of nitric oxide (NO) and a potent S‐nitrosating agent which reduced histologic brain injury after rat asphyxial cardiac arrest (ACA). The mechanism(s) of nitrite‐mediated neuroprotection remain to be defined. We hypothesized that nitrite‐mediated brain mitochondrial S‐nitrosation accounts for neuroprotection by reducing reperfusion reactive oxygen species (ROS) generation. Nitrite (4 μmol) or placebo was infused IV after normothermic (37°C) ACA in randomized, blinded fashion with evaluation of neurologic function, survival, brain mitochondrial function, and ROS. Blood and CSF nitrite were quantified using reductive chemiluminescence and S‐nitrosation by biotin switch. Direct neuroprotection was verified in vitro after 1 and 4 h neuronal oxygen glucose deprivation measuring neuronal death with inhibition studies to examine mechanism. Mitochondrial ROS generation was quantified by live neuronal imaging using mitoSOX. Nitrite significantly reduced neurologic disability after ACA. ROS generation was reduced in brain mitochondria from nitrite‐ versus placebo‐treated rats after ACA with congruent preservation of brain ascorbate and reduction of ROS in brain sections using immuno‐spin trapping. ATP generation was maintained with nitrite up to 24 h after ACA. Nitrite rapidly entered CSF and increased brain mitochondrial S‐nitrosation. Nitrite reduced in vitro mitochondrial superoxide generation and improved survival of neurons after oxygen glucose deprivation. Protection was maintained with inhibition of soluble guanylate cyclase but lost with NO scavenging and ultraviolet irradiation. Nitrite therapy results in direct neuroprotection from ACA mediated by reductions in brain mitochondrial ROS in association with protein S‐nitrosation. Neuroprotection is dependent on NO and S‐nitrosothiol generation, not soluble guanylate cyclase.

Global assessment of oxidized free fatty acids in brain reveals an enzymatic predominance to oxidative signaling after trauma

Traumatic brain injury (TBI) is a major health problem associated with significant morbidity and mortality. The pathophysiology of TBI is complex involving signaling through multiple cascades, including lipid peroxidation. Oxidized free fatty acids, a prominent product of lipid peroxidation, are potent cellular mediators involved in induction and resolution of inflammation and modulation of vasomotor tone. While previous studies have assessed lipid peroxidation after TBI, to our knowledge no studies have used a systematic approach to quantify the global oxidative changes in free fatty acids. In this study, we identified and quantified 244 free fatty acid oxidation products using a newly developed global liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. This methodology was used to follow the time course of these lipid species in the contusional cortex of our pediatric rat model of TBI. We show that oxidation peaked at 1h after controlled cortical impact and was progressively attenuated at 4 and 24h time points. While enzymatic and non-enzymatic pathways were activated at 1h post-TBI, enzymatic lipid peroxidation was the predominant mechanism with 15-lipoxygenase (LOX) contributing to the majority of total oxidized fatty acid content. Pro-inflammatory lipid mediators were significantly increased at 1 and 4h after TBI with return to basal levels by 24h. Anti-inflammatory lipid mediators remained significantly increased across all three time points, indicating an elevated and sustained anti-inflammatory response following TBI.

Aiming for the target: Mitochondrial drug delivery in traumatic brain injury

ABSTRACT Mitochondria are a keystone of neuronal function, serving a dual role as sustainer of life and harbinger of death. While mitochondria are indispensable for energy production, a dysregulated mitochondrial network can spell doom for both neurons and the functions they provide. Traumatic brain injury (TBI) is a complex and biphasic injury, often affecting children and young adults. The primary pathological mechanism of TBI is mechanical, too rapid to be mitigated by anything but prevention. However, the secondary injury of TBI evolves over hours and days after the initial insult providing a window of opportunity for intervention. As a nexus point of both survival and death during this second phase, targeting mitochondrial pathology in TBI has long been an attractive strategy. Often these attempts are mired by efficacy‐limiting unintended off‐target effects. Specific delivery to and enrichment of therapeutics at their submitochondrial site of action can reduce deleterious effects and increase potency. Mitochondrial drug localization is accomplished using (1) the mitochondrial membrane potential, (2) affinity of a carrier to mitochondria‐specific components (e.g. lipids), (3) piggybacking on the cells own mitochondria trafficking systems, or (4) nanoparticle‐based approaches. In this review, we briefly consider the mitochondrial delivery strategies and drug targets that illustrate the promise of these mitochondria‐specific approaches in the design of TBI pharmacotherapy. This article is part of the Special Issue entitled “Novel Treatments for Traumatic Brain Injury”. HIGHLIGHTSTBI prompts mitochondrial dysfunction; no clinically effective therapies exist.Generalizable strategies allow specific drug targeting of mitochondrial pathology.Colocalization therapy with its target may improve potency and specificity.Mitochondrial‐localization can improve new and old therapies in TBI.

Oxidized phospholipid signaling in traumatic brain injury

Abstract Oxidative stress is a major contributor to secondary injury signaling cascades following traumatic brain injury (TBI). The role of lipid peroxidation in the pathophysiology of a traumatic insult to neural tissue is increasingly recognized. As the methods to quantify lipid peroxidation have gradually improved, so has the understanding of mechanistic details of lipid peroxidation and related signaling events in the injury pathogenesis. While free‐radical mediated, non‐enzymatic lipid peroxidation has long been studied, recent advances in redox lipidomics have demonstrated the significant contribution of enzymatic lipid peroxidation to TBI pathogenesis. Complex interactions between inflammation, phospholipid peroxidation, and hydrolysis define the engagement of different cell death programs and the severity of injury and outcome. This review focuses on enzymatic phospholipid peroxidation after TBI, including the mechanism of production, signaling roles in secondary injury pathology, and temporal course of production with respect to inflammatory response. In light of the newly identified phospholipid oxidation mechanisms, we also discuss possible therapeutic targets to improve neurocognitive outcome after TBI. Finally, we discuss current limitations in identifying oxidized phospholipids and possible methodologic improvements that can offer a deeper insight into the region‐specific distribution and subcellular localization of phospholipid oxidation after TBI. Graphical abstract Figure. No Caption available.

Abstract: A Qualitative Survey Study of United States Burn Units

CONCLUSION: The study suggests the closed-loop technique significantly decreases time of fat transfer to an average of 15 minutes, a trend towards decreased overall complications, though not statistically significant, and good subjective fat retention beyond 12 months. This suggests the closed-loop method is safe, effective, and significantly decreases operating time. We continue to collect patients in the closed-loop cohort and are also analyzing variation in adipocyte viability between the two methods.

Disentangling oxidation/hydrolysis reactions of brain mitochondrial cardiolipins in pathogenesis of traumatic injury

Mechanical injury to the brain triggers multiple biochemical events whose specific contributions to the pathogenesis define clinical manifestations and the overall outcome. Among many factors, mitochondrial injury has recently attracted much attention due to the importance of the organelle for bioenergetics as well as intra- and extracellular signaling and cell death. Assuming the essentiality of a mitochondria-unique phospholipid, cardiolipin (CL), for the structural and functional organization of mitochondria, here we applied global (phospho) lipidomics and redox lipidomics to reveal and identify CL modifications during controlled cortical impact (CCI). We revealed 2 major pathways activated in the CCI-injured brain as time-specific responses: early accumulation of oxidized CL (CLox) products was followed by hydrolytic reactions yielding monolyso-CLs (mCLs) and free fatty acids. To quantitatively assess possible specific roles of peroxidation and hydrolysis of mitochondrial CL, we performed comparative studies of CL modifications using an animal model of Barth syndrome where deficiency of CL reacylation (Tafazzin [Taz] deficiency) was associated exclusively with the accumulation of mCLs (but not CLox). By comparing the in vitro and in vivo results with genetic manipulation of major CL-, CLox-, and mCL-metabolizing enzymes, calcium-independent phospholipase A2γ and Taz, we concluded that the 2 processes - CL oxidation and CL hydrolysis - act as mutually synergistically enhancing components of the pathogenic mechanism of mitochondrial injury in traumatic brain injury. This emphasizes the need for combined therapeutic approaches preventing the formation of both CLox and mCL.